Cell free DNA (“cfDNA”) can be analyzed to provide a prognosis, diagnosis or a prediction of a response to a treatment for a variety of diseases and conditions, including various cancers, transplant failure or success, inflammatory diseases, infectious disease and fetal aneuploidy.
Cell-free fetal DNA (cffDNA) is present in the blood of a pregnant female. This discovery led to the possibility of performing non-invasive prenatal testing (NIPT) of a fetus using a blood sample from the pregnant female. Invasive prenatal tests (e.g., amniocentesis or chorionic villi sampling (CVS)) can be stressful for the mother and some believe such procedures may increase the risk of miscarriage. NIPT can provide information related to a variety of genetic defects, including Down syndrome (trisomy chromosome 21), Patau syndrome (trisomy 13), and Edwards syndrome (trisomy 18). Such methods should be highly robust as a false positive may lead to unnecessary medical procedures, and a false negative may deprive the expectant mother of understanding the available medical options.
There are many technical hurdles associated with implementing a non-invasive prenatal test on a clinical scale. For example, many NIPT efforts have focused on the analysis of cffDNA to identify copy number changes in particular sequences (e.g., sequences from chromosome 21). However, such methods are difficult to implement in a robust way because, in part, the vast majority of cfDNA in a blood sample is maternal in origin and in many cases only a very small amount (e.g., on average ˜10% and down to about 3%) is from the fetus. For example, the presence or absence of an extra copy of a chromosome (such as chromosome 21) in the fetus may be determined by comparing the copy number of sequences corresponding to chromosome 21 to the copy number of sequences corresponding to an autosomal chromosome. While such methods sound attractive, they are in fact challenging because the fractional concentration of fetal DNA relative to maternal DNA in maternal blood can be as low as 3%. As such, for every 1000 sequences corresponding chromosome 21 that are in the maternal bloodstream, only a small percentage of those sequences (e.g., 30 sequences if the fetal fraction is 3%) are from the fetus. Thus, an extra copy of a chromosome in the fetus will only lead to a relatively small increase in the number of sequences corresponding to that chromosome in the maternal bloodstream. For example, if the fetal fraction is 4, fetal trisomy 21 will only lead to a 1.5% increase in the number of fragments corresponding to chromosome 21 in the maternal bloodstream. As a result of this problem, statistical rigor can only be achieved by counting large numbers of sequences corresponding to a chromosomal region that is suspected of having a copy number difference (e.g., at least 1,000 and sometimes at least 5,000 or more sequences) and comparing that number to a similar number for another chromosomal region that is not suspected of having a copy number difference. Being able to consistently and accurately count fragments is paramount to the success of many NIPT methods.
Some NIPT methods use polymerase chain reaction (PCR) to amplify the DNA. PCR is widely used, but it suffers from various limitations that can negatively affect the accuracy of the results. PCR can introduce sequence artifacts and create amplification bias in a sample. PCR sequence artifacts are errors introduced into the DNA sequence of the PCR amplified product by the PCR reaction. PCR sequence artifacts can be caused by various events, such as by the formation of chimeric molecules (e.g., two different pieces of DNA joined end to end), the formation of heteroduplex DNA (e.g., the hybridization of two different DNA molecules to each other) and by errors made by the amplification enzyme (e.g., by Taq DNA polymerase placing a mismatched nucleotide onto the DNA template). Sequence bias from PCR is a skewing of the distribution of PCR products compared to the original sample. PCR sequence bias can be caused by various events, such as intrinsic differences in the amplification efficiency of templates or inhibition of amplification due to self-annealing of DNA templates. PCR errors result in an unequal amplification of the different DNA molecules so that the amplified sample is no longer representative of the original sample. PCR is also notoriously sensitive to exogenous DNA contamination from the environment. Due to the exponential amplification of DNA during PCR, even very small amounts of exogenous DNA contamination in a PCR reaction can lead to highly inaccurate results. Exogenous DNA contamination can be introduced from aerosolized droplets floating in the air or can be transferred into a reaction from contaminated equipment.
Use of rolling-circle amplification (RCA) to analyze cfDNA in maternal blood avoids many of the problems associated with PCR. However, RCA products are not very easy to quantify in a way that provides statistical robustness. At a practical level, although the absolute numbers of products in an RCA reaction may be sufficiently high to provide statistical robustness, different RCA products may be amplified and detected at different efficiencies and, as such, consistently detecting tens or hundreds of thousands of RCA products evenly has been challenging.